THE  FILAMENTOUS  BACTERIA  OF  ACTIVATED  SLUDGE 


HARVEY  LAWRENCE  LONG 


THESIS 

FOR  THE 

DEGREE  OF  BACHELOR  OF  ARTS 

IN 

BOTANY 


COLLEGE  OF  LIBERAL  ARTS  AND  SCIENCES 
UNIVERSITY  OF  ILLINOIS 


1922 


/ 922 
L85I 


UNIVERSITY  OF  ILLINOIS 


.May. 24 1 92. 2.  _ 


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ENTITLED T _he_  ^ e nt  o_u  s_  _B_a  c_t  e r_i  a _ of  _ Ac  t_i  y a.  t ed_  _S  lu  dge 


IS  APPROVED  BY  ME  AS  FULFILLING  THIS  PART  OF  THE  REQUIREMENTS  FOR  THE 


DEGREE  OF  


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TABLE  OF  CONTENTS 


Page 

I.  Introduction , 1 

II.  Historical 

1.  The  Biology  of  Various  Methods  of 

Sewage  Disposal 2 

2.  A Review  of  the  Taxonomy  of  the  Chlamedo- 

bacteriaceae ...... 4 

3.  A Review  of  the  Taxonomy  of  the  Beggiato- 

aceae 18 

III.  Material  and  Methods .22 

IV.  Observations , . 23 

V.  Summary , 31 

VI.  Acknowledgement 32 

VII.  Bibliography  .....  33 

VIII.  Plates  and  Descriptions .34 


THE  FILAMENTOUS  BACTERIA  OF  THE  ACTIVATED  SLUDGE 
PROCESS  OF  SEWAGE  DISPOSAL. 

By 

Harvey  L.  Long 
I 

INTRODUCTION 

The  activated  sludge  process  of  sewage  disposal  is 
said  to  have  had  its  inception  at  Lawrence, Mass. , in  1912. 

In  "The  44th  Annual  Report,  State  Board  of  Health  of  Mass., 
1912"  (p.275-367)  a sub-chapter  (p.  2S0-2S2)  records  the 
results  of  an  experiment  on  the  aeration  of  sewage  as  an  aid 
to  filtration  (Porter,  p.  7,  sec.  1). 

Previous  observations  on  aeration  led  to  a series  of 
experiments  begun  in  April  1912,  to  test  the  efficiency  of 
aeration.  The  sides  of  a slate  tank  became  covered  with  a 
brown  growth  which  removed  the  suspended  matter  and  a large 
portion  of  the  colloids  from  the  sewage  after  a few  hours’ 
treatment.  Experiments  were  continued  at  the  Lawrence  (Mass.1) 
experimental  station,  and,  at  the  present  time,  the  principal 
sewage  studies  there  are  concerned  with  the  activated  sludge 
process. 

Numerous  municipal  and  private  interests  have  been 
conducting  experiments  with  the  activated  sludge  process  since 


2 

that  time.  The  earlier  municipal  experimental  plants  are  at 
Milwaukee  (1912),  Houston  (1915),  Cleveland  (1915),  Worcester, 
England  (1915),  Sheffield, England  (1915).  Several  industrial 
plants  have  conducted  experiments  on  the  disposal  of  industrial 
wastes  by  the  activated  sludge  process.  More  recently  (1920) 
the  Chicago  Sanitary  District  has  conducted  experimental  and 
semi- exper imental  plants  (Porter,  Indexes).  Since  1914,  the 
Illinois  State  Water  Survey  at  the  University  of  Illinois  has 
maintained  what  Porter  (p.106)  believes  to  be  the  first  exclusive 
activated  sludge  plant  in  the  United  States.  Studies  on  that 
plant  have  been  conducted  along  many  lines. 

II 

HISTORICAL 

1.  The  Biology  of  Various  Methods  of  Sewage  Disposal. 

Certain  investigations  have  been  carried  out  upon  the 
flora  and  fauna  of  the  filters.  In  general,  a very  wide  range 
of  life  is  represented.  Dr.  Parkinson  (1919)  at  the  University 
of  Birmingham  found  many  algae  and  definitely  isolated  the 
following  from  the  slimy  growth  on  filters:  Stichococcus, 

Chlor ella.  Scenedesmus . Nitzschia,  Navicula.  Oscillator ia.  and 
Phormidlum.  The  most  common  insect  found  was  the  "Spring  Tail" 
Aohorutes.  J.W.  Haigh  Johnson  (1914)  found  the  dominating 
or ganisrr^'on  sewage  filters  to  be  (a)  Flies,  (b)  Achorutes,  (c) 
Bacillus  colon  and  Thiothrlx  nivea.  In  the  upper  layer  of 
the  filter  bed  he  lists  the  following  organisms:  Zoo gloea 

rami ?;era,  Sphaerotilus  natans.  Begyiatoa  alba.  Thiothrlx  nivea  , 


3 


Chromatium  okenii.  Polytoma  uvela,  Saprolegnla,  Hyphae  of 
Mucors . Lower  down  in  the  filter  bed,  the  Zoogloeal  masses 

were  fewer  and  Rotifers,  Ciliates,  Nematodes,  and  aquatic 
earthworms  abounded. 

In  the  study  of  the  biology  of  the  sprinkling  filter 
( N. J.  Ag. Ex ten. Bull  No.  352),  a gradual  change  in  flora 
and  fauna  from  top  downward  was  observed,  the  greatest  activity 
being  in  the  center  of  the  bed. 

Cox  (.1921),  in  discussing  the  no n-bacterial  population 
of  the  sewage  trickling  filter,  reports  that  the  types  of 
organisms  are  very  limited.  He  made  ho  attempt  to  determine 
the  species  or  family  in  some  cases  "because  little  practical 
importance  is  attached  to  detailed  biological  data".  He  lists 
three  common  algae,  the  same  number  of  protozoa,  and  a few 
worms  and  insects.  Schi zomycetes  found  were  Zoogloea  . Spjrell a. 
Cr  eno thrix,  and  Beg^iatoa. 

There  have  been  a few  studies  made  of  the  biology  of 
the  activated  sludge,  but  it  seems  that  all  have  neglected  the 
most  prominent  organisms  in  the  sludge,  the  filamentous  forms. 

Hommon  (1918,  p.45),  in  his  studies  on  the  treatment 
and  disposal  of  industrial  wastes,  ran  a test  with  activated 
sludge.  His  experimental  chambers  were  an  inch  in  diameter  and 
four  feet  long.  He  made  counts  of  the  animal  organisms  present 
and  he  lists  several  Ciliates,  Flagellates,  Rotifers  and  molds. 

He  records  zoogloeal  masses  in  standard  units,  the  highest  being 
255,000  per  cc. 


In  a series  of  articles,  Paul  Courmont  and  co-workers 


4 


present  the  results  of  an  investigation  of  the  bacterial  flora 
of  sewage  purified  by  activated  sludge.  Their  study,  however, 
is  confined  to  the  determination  of  the  reduction  in  count  in 
the  effluent  of  forms  that  grow  on  the  different  media. 

Russell  (1916,  p.354)  made  a study  of  the  bacterial 
flora  of  sewage  purification  by  aeration  in  the  laboratories  of 
the  State  Water  Survey  of  Illinois.  Again  no  attention  was  paid 
to  the  filamentous  forms  present.  Observations  were  made  only 
on  the  organisms  that  would  grow  on  the  ordinary  media.  In  as 
much  as  filamentous  forms  do  not  grow  on  the  media  used,  they 
were  not  observed. 

2.  A Review  of  the  Taxonomy  of  the  Chlamedobacteri aceae 

The  Chlamedobacteri aceae  . a group  of  filamentous 
bacteria,  have  long  been  called  iron  bacteria  because  of  the 
occasional  occurence  of  iron  oxide  in  connection  with  the 
organisms.  The  deposition  of  iron  is  not  common  to  all  members 
of  the  group.  In  certain  forms  the  deposition  of  iron  oxide 
in  connection  with  the  filaments  is  the  usual  thing,  in  others 
occasional,  and  in  others  never.  Furthermore,  there  is  not  yet 
a technique  developed  for  the  obtaining  of  the  filamentous 
forms  in  pure  culture.  The  classification  of  these  organisms 
is  based  on  the  original  and  only  method  remaining,  the  morpho- 
logical basis. 

At  present,  there  is  considerable  variation  in  the 
description  of  the  respective  forms  and  considerable  uncertainty 
as  to  the  tenability  of  certain  genera  and  species.  Although  it 


5 


is  not  the  task  of  this  paper  to  determine  taxonomic  questions, 
it  will  be  necessary,  because  of  the  mentioned  uncertainty, 
to  define  the  basis  assumed  in  the  determination  of  the  particu- 
lar forms  present  in  the  activated  sludge.  Because  of 
variations  in  descriptions,  because  of  the  scattered  sources 
of  material,  because  much  of  the  original  literature  is  not 
in  English,  and  because,  in  order  to  identify  the  filamentous 
forms,  the  writer  has  had  to  spend  months  reviewing  literature 
on  the  subject,  he  has  chosen  to  include  the  essential  points 
which  are  concerned  in  identification, 

Ellis  has  contributed  perhaps  the  greater  part  of 
the  literature  and  added  a considerable  fund  of  new  information 
during  the  last  quarter  century,  Many  of  his  writings  will 
be  referred  to  in  their  original  source,  but  recently  much  of 
his  published  work  and  some  of  the  unpublished  were  gathered 
together  in  one  volume,  "The  Iron  Bacteria”,  published  by  the 
Frederick  A.  Stokes  Co.,  New  York,  bearing  no  date  of  publication. 
(Elli3,  a). 

One  of  the  most  recent  reports  of  studies  of  the 
literature  of  the  Chlarnedobacteriales  is  that  of  Buchanan  (1916 
a,  p.301),  in  which  he  records  one  family  and  five  genera. 
Following  is  Buchanan’s  list  including  an  additional  genus  - 3. 

Order  II.  Chlarnedobacteriales.  (Or do  nov.)  Buchanan 

(a)  1913  p.301) 

Family  I.  Chlamedobacter iaceae,  Migula,  1394, p,  237 


Genus 

1. 

Leotothrix,  Kuet zing, 1843,  p. 

198 

Genus 

2. 

Didymohelix, 

Griffith,  1353, 

p.  438 

Genus 

3. 

Spirophyllum, 

Ellis,  1907, (a) 

p.  21 

6 


Genus 

4. 

Creno thrix. 

Cohn,  1370,  p.  130 

Genus 

5. 

Sphaero  tilus 

, Kuetzing,  1833,  p.385 

Genus 

6. 

Clono thrix. 

Schorler,  1904,  p.689 

Buchanan  describes  (1918,  (a)p.30l),  the  Chlamedo- 
bacteriaceae  as  filamentous  bacteria,  typically  water  forms, 
frequently  sheathed,  without  true  brahching,  although  false 
branching  may  be  present.  The  sheath  is  frequently  impregnated 
with  iron.  Conidia  may  be  developed,  but  never  endospores. 
Sulphur  granules  or  bacter iopurpurin  never  present.  Mature 
cells  or  filaments  neither  motile,  nor  protozoan-like. 

Migula’s  description  (1900,  p.  1030)  follows.  Cells 
are  cylindrical,  and  arranged  in  a thread  with  a sheath. 
Reproduction  takes  place  through  conidia  which  do  not  pass 
through  a resting  period  but  develop  immediately. 

Genus  LEPTOTHRIX  (Kuetzing,  1843,  p.198)  emend.  Buchanan,  1913. 

Synonyms: 

Chlamsdo thrix  Migula,  1900,  p.1030 

Lepto trichia  Trevisan,  1879,  p.  929 

Detoniella  Trevisan,  1885,  p.  929 

The  following  description  given  by  Buchanan  embraces 
the  important  characters  of  the  genus.  "Filaments  of  color- 
less cylindrical  cells,  later  thicker,  yellortr'or  brown, 
becoming  incrusted  with  iron  oxide.  The  iron  may  be  dissolved 

by  dilute  acid,  where  upon  the  cells  show  up  well.  Multipli- 
cation i3  through  the  division  and  abstriction  of  cells,  and 


7 


motile  cylindrical  swarm  cells.  Swarm  cells  sometimes  germinate 
in  the  sheath  and  give  the  appearance  of  branching.  Pseudo- 
dichotomous  branching  may  occur." 

This  genus  has  been  re-described  by  two  men  previous 
to  Buchanan:  by  Cohn  (1S75,  p.  203)  and  by  Ellis  (1907,  (b) 
p.503). 

Number  of  species,  five. 

In  as  much  as  Leptothrix  ochraceae  is  the  only  one 
that  is  sufficiently  described  for  practical  purposes,  the 
discussion  will  be  confined  to  that  species. 

Leptothrix  Qchraceae  Kuetzing,  1843,  p.  198 

According  to  Ellis,  L.  Ochracea  is  by  far  the  most 
widely  distributed  of  all  of  the  iron  bacteria,  occuring  in  all 
parts  of  the  world.  It  occurs  in  most  iron  bearing  waters. 

Very  little  is  known  of  the  internal  structure  of  the 
filament.  In  nature  it  becomes  encrusted  and  is  easily 
recognized  because  of  the  permanent  tubular  nature  of  the  encrusted 
sheath  with  its  distinctly  ochraceou3  or  yellow-red  coloring. 

The  average  thread  measures  1 1/2  to  2 micron,  but  it 
may  increase  to  3 and  reach  a length  of  200  micron.  Plates 
showing  Leptothrix  ochracea  are  to  be  found  opposite  page  2, 

Ellis,  "The  Iron  Bacteria". 

The  writer  has  found  young  filaments  in  the  fuscous 
colored  growths  on  water  faucets  which  are  kept  moist  constantly. 

In  these  young  filaments  the  cellular  nature  of  the  filament 
inside  the  sheath  was  quite  discernable  without  staining.  The 
cells  were  from  1 to  1.5  microns  broad  and  from  two  to  thr ee 


8 

times  as  long. 

Genus  DIDYMOHELIX  Griffith,  1853,  p.  438 

Synonyms  according  to  Buchanan  in  part. 

Galionella  Bory,  1823,  in  part 
Gallionella  Ehreriberg,  1838,  p.  166,  in  part. 
Gloeotila  Kutzing,  1843,  p.245,  in  part 
Species,  one  broad  species  unless  Spirophyllum  be 
included  in  the  genus. 

Didymohelix  ferru^inea,  (Ehr)  Griffith,  1353 
The  filaments  are  twisted,  simple,  or  two  filaments 
twisted  together.  Young  cells  colorless,  later  yellow-brown 
to  rust  red,  through  the  deposition  of  iron.  Simple  filaments 
show  no  division  into  cells.  Sheath  not  demonstrable,  (Buchanan 
1913  a,  p.  304). 

Ellis  compares  the  structure  (a.  p.19)  to  the 
twisting  of  a wire  hairpin  spirally  on  itself.  Average  thickness 
of  that  thread  is  1/2  micron  to  3/4,  although  it  may  reach 
1 1/2  micron.  The  loops  formed  by  the  twisting  thread  may  b e 
from  2 to  6 microns  in  amplitude. 

The  writer  has  found  Didymohelix  growing  on  water 
faucets  of  the  University  water  supply. 

What  are  the  phylogenetic  relations  of  Spirophyllum 
f errugineum  Ellis  and  Uodophvllum  ferrugineum  Ellis  with 
each  other,  and  with  Didymohelix  ferru^inea  Griffith?  Buchanan 
(1318,  (a),  p.  304)  left  that  a question.  The  writer  can  see 

no  particular  likeness,  morphologically,  between  the  latter  and 


« 


9 


the  two  former.  However,  from  the  description  that  will 
follow,  it  will  be  seen  that  the  two  forms  first  named  have 
some  morphological  characteristics  in  common  and  might  be 
included  in  one  genus. 

Genus  SPIROPHYLLUM  Ellis,  1907,  ( (a),  p.  21  (b)  p.  507) 

Type  species  (monotyp.)  - S.  ferrugineum.  Body  of 
cell  elongated,  flattened,  and  spirally  twisted.  Number  of 
spiral  turns  may  vary  from  a quarter  turn  to  fifteen  or  more. 
Width  varies  from  1 to  6 microns,  length  may  reach  200  micron. 

No  definite  membrane  but  edge  is  thickened  so  as  to  form  a 
sort  of  rampart  all  around  the  cell.  Multiplication  by  the 
formation  of  conidia,  by  external  constriction,  which  are  oval. 
Conidia  are  formed  before  twisting  begins.  Ferric  hydroxide 
deposited  on  the  surface.  Found  only  in  iron  water,  and 
always  in  connection  with  Lentothrix  ochracea,  when  found  at  all. 
(Enlows,  1920,  p.  86). 

Nodophyllum  ferrugineum  is  also  flattened,  but 
restricted  at  intervals,  being  arched  rather  than  spirally 
twisted.  Otherwise  it  is  quite  similar  to  Soirophvllum  and 
deserves  the  same  generic  name.  But,  in  as  much  as  there 
have  been  no  recorded  observations  of  Nodophyllum  since  the 
original,  perhaps  there  should  be  no  definite  decision  as  to 
their  position  until  further  study  has  been  made. 

It  is  apparent  that  in  many  cases,  as  shall  be 
noted  later,  new  genera  have  been  created  unnecessarily. 


Genus  CRENOTHRIX. 


10 

Cohn,  1870,  p.  130 

Synonyms  according  to  Ellis  (a,  p.  41) 

Leptothrix  kuhniana  R ab  enho r s t 

Cr  eno t hr i x kuh n i ana  Zopf 

Crenothrix  manganifera  Jackson 
Leptothrix  meyeri  Ellis 

Megalothrix  discophora  Schwers 

Number  of  species,  one. 

Crenothrix  polyspora.  Cohn 

Following  is  Migula' s (190C,  p.  1033)  description 
of  Crenothrix. 

Non-branching  thread  bacteria,  with  contrast  of 
base  and  tip.  Attached,  with  a thickening  of  the  free  end. 
Sheath  moderately  thick,  and  encrusted  with  iron  oxide  if  in 
iron  bearing  water.  Cells  cylindrical  to  disc-like.  Reproduce 
through  spherical  non-mo  tile  conidia  formed  from  the  vegetative 
cells  which  divide  in  three  planes  and  cause  the  expansion  of 
the  tip  of  the  filament.  The  conidia  become  free  and  germinate 
often  on  the  sheath  of  the  mother  filament. 

Filaments  vary  from  1.5  to  5 microns  at  the  base; 
from  4 to  9 at  the  tip.  Filaments  may  reach  a length  of  2 to 
3 mm.  Sheath  on  the  thin  threads  is  very  delicate.  Young 
threads  very  often  show  no  sheath  at  the  tip.  Threads  usually 
vary  in  thickness. 

Cr  eno thrix  is  very  widely  distributed  (Ellis,  a,  p. 
43).  It  occurs  in  brooks,  drain  pipes,  water  pipes,  and  in 


u 


« 


u 

small  quantities,  in  open  waters.  It  is  present  perhaps  in  a 
large  percentage  of  ferruginous  water. 

in  addition  to  the  description  given  by  Migula,  Ellis 
(a,  p.  49)  gives  some  additional  facts  from  his  observations. 

Growth  takes  place  by  fission,  and,  as  the  mucilaginous  sheath 
hardens,  continued  division  and  growth  break  the  septa  of  the 
now  fixed  sheath,  and  a tube  results.  In  this  manner,  large 
cells  may  be  forced  from  the  end  of  the  filament  as  macrogonidia. 

It  often  happens  that  the  or ganism retains  throughout 
life  the  cylindrical  form  of  its  immature  stage,  and  that  it  does 
not  assume  the  trumpet  shape  customarily  assumed  by  the  adult 
organisms. 

An  important  difference  between  Creno thrix  and  Sphaero tilus 
cladothrix  is  that  the  cells  of  the  former  are  visible  without 
staining.  A plate  showing  a young  filament  of  Cr eno thrix.  5 
microns  in  diameter,  is  shown  opposite  page  42,  Ellis,  The  Iron 
Bacteria. 

Cohn  (1870,  p.  130),  in  the  original  description,  gives 
the  size  of  filaments  1.5  to  5.2  microns  broad;  vegetative  cells 
in  length  are  1/2  to  4 times  the  breadth  of  the  filaments.  They 
occur  in  stagnant  and  running  water  containing  organic  matter  and 
iron  salts. 

Benecke  (1912,  p.  491)  gives  the  size  of  the  micrcgonidia 
(the  spherical  spores  formed  as  the  result  of  the  division  of  the 
spiral  cells  into  three  planes'!  as  2 to  4 microns  in  diameter,  and 
the  macrogonidia  may  reach  a length  of  7 microns. 

Jackson  (1902)  gives  the  size  of  the  vegetative  cells 


-L  lj 

varying  from  1.5  microns  in  width  to  2 to 5 microns  in  length. 
Schorler  (1904,  p.  691)  says  that  the  cells  are  twice  as 
long  as  wide. 

Summary  of  Crenothrix 

Non-br  anching  thread  bacteria,  which  may  or  may 
not  have  contrast  in  base  and  tip.  Sheath  delicate,  moderately 
thick  until  encrusted  with  iron  oxide.  Cells  of  the  filament 
may  vary  in  thickness;  in  length  from  cylindrical  to  discoidal; 
in  size  1.5  to  2 micron  wide  by  2 to  7 microns  in  length,  in 
general,  twice  as  long  as  wide.  Cells  visible  without  stain- 
ing. Growth  by  fission.  Reproduction  by  spherical  microgonidia, 
2 to  4 microns  in  diameter,  as  a result  of  division  of  vegeta- 
tive cell  in  three  planes.  Also  by  macro  go nidi a,  escaped  or 
isolated  vegetative  cells.  Occurs  in  stagnant  or  running 
water  containing  organic  matter. 

Genus  SPHAEROTILUS:  (Kutzing,  1333)  emend.  Buchanan,  1913 

Synonyms; 

Cl  ado thr ix  Cohn,  1875,  p.  185 

Chlamedo thrix  Molish,  1910 
Following  is  a summary  of  Buchanan’s  description  of 
the  genus  (1918,  1,  p.  305):  Filaments  of  rods  or  oval  cells, 
attached,  colorless,  showing  pseudodi cho tomous  or  false 
branching;  multiplication  by  motile  swarm  cells  and  non-mo  tile 
conidia,  the  former  with  a clump  of  flagella  near  one  end. 

Number  of  species  described,  two: 

S.  natans  Kutzing,  1833  (p.  385) 

(Cohn)  era.  Migula  ( 1900, p . 1035) 


S.  dichotoraus 


13 


For  some  time  this  genus  has  been  designated  by  the 
name  Cl ado thr ix . but  the  generic  name  Clado thrix  belongs  to 
the  Hyphomyoeten  and  above  all  not  to  the  bacteria  (Migula,  1900 , 
p.  1036).  Undoubtedly  the  species  that  Cohn  called  Clado  thrix 
is  more  nearly  related  to  S.  natans  than  Creno thrix  polyspor a , 
but,  as  shall  be  observed  later,  Creno thrix  and  S , natans  are 
sufficiently  similar,  on  the  present  basis  of  classification 
to  be  considered  species  of  the  same  genera,  if  not  varieties 
of  the  same  species  or  variants  of  a common  type. 

Some  have  thought  (Benecke,  1912,  p.  492),  because 
members  of  the  genus  Sphaero tilus  do  not  cause  deposition  of 
iron  oxide,  that  the  fact  was  sufficient  to  separate  this  genus 
from  others.  But  Creno  thr  ix  is  not  always  iron  incrusting  and 
so  separation  on  that  premise  is  untenable. 

Variation  of  environment  influences  not  only  the 
morphology  of  these  forms  but  also  the  physiology,  as  shall  be 
noted  later,  a fact  that  has  not  been  appreciated  by  former 
investigators.  Ellis  advises  retaining  the  name  Clado  thrix 
for  this  genus  (a,  p.92),  evidently  because  it  is  difficult  to 
abandon  the  familiar  name  for  the  unfamiliar. 

Sphaero tilus  natans  Kutzing,  1833 

Following  is  Migula' s description  of  this  species. 
(1900,  p.  1035). 

The  organism  will  be  found  in  yellowish-brown  "floes" 
at  times  free  floating  in  dirty  water,  which  under  the  microscope 
are  composed  of  bundles  of  colorless  threads.  The  threads  are 

composed  of  cylindrical,  somewhat  rounded  cells,  which  in  the 


14 


younger  threads  only  are  close  together,  while  in  the  older 
there  are  wider  intervals  between  the  cells. 

Altogether,  the  cells  of  S.  natans  are  very  much  like 
S.  dichotomus  as  thick  (2  microns , but  seldom  so  pressed 
together.  The  sheath,  opposed  to  that  of  dichotomus.  is 
extremely  delicate,  slimy  and  very  difficult  to  see.  (This  does 
not  mean  thin,  for  the  sheath  of  S.  natans  is  thicker  but  less 
visible) . 

Eidam  found,  throughout  the  homogenous  protoplasm, 
exceedingly  numerous,  small  spherical  parts,  which,  in  sharp 
definition  showed  strong  refraction  in  every  separate  cell 
(1876,  p.  133).  Every  cell  is  a sporangium  and  a multitude  of 
small  round  spores.  The  spores  germinate  very  soon  and  form  thin 
delicate  filaments  to  which,  when  the  mother  filament  is  present, 
they  become  attached.  More  and  more  of  the  spores  develop 
sporelings,  until  the  slowly  decomposing  mother  filament  becomes 
thickly  surrounded  with  the  young  sporelings. 

Migula  in  all  of  his  observations  never  saw  this 
state  of  affairs.  This  phenomenon  was  of  common  occurrence  in 
the  studies  made  by  the  writer  of  the  flora  of  the  activated 
sludge,  and  it  remains  to  be  determined  whether  Eidam  really 
had  Sphaero tilus  or  whether  it  was  a new  or  allied  form  (see 
Plate  I,  Figs.  1 and  2). 

Eidam  is  the  only  one  to  describe  a member  of  the 
Chi amedobacteriaceae  as  having  endospores. 

J.W.Haigh  Johnson  (1914,  p.121)  gives  the  following 
dimensions  for  Sphaero tilus  natans.  Filaments,  6 to  8 microns 


thick,  with  cells  2 microns  wide  by  4 to  6 microns  long. 


. 

. 

15 


Gelatinous  sheath  thick  but  not  readily  visible.  Multiplication 
by  transverse  cell  division  or  fission  also  by  swarm  cells.  Can 
be  found  in  almost  every  stream  receiving  unpurified  sewage, 
also  in  the  effluent  of  industrial  wastes. 

Zoogloea  form  is  often  assumed  by  S.  natans.  These 
gelatinous  masses,  called  Zoogloea  ramiaera.  1000  to  2000  by 
15  microns  in  size,  contain  rod-like  cells  1 micron  thick  im- 
bedded within  the  matrix.  See  Plate  IV,  Fig.  7 for  a type  of 
Zoogloea  ramigera. 

A habit  growth  of  S.  natans  is  in  many  parallel 
strands  with  a common  mucilaginous  sheath  (Benecke,  p.203). 

Ellis  (a)  also  makes  the  same  observation,  S.  natans  closely 
resembles  S.  dichotoma  but  differs  from  it  in  that  the  threads 
run  parallel,  all  being  enveloped  by  a common  mucilaginous 
cover  ing. 

Summary  of  Sphaerotilus  nutans 
Parallel  filaments,  enveloped  in  a more  or  less 
common  mucilaginous  covering.  Filaments  attached,  colorless, 
composed  of  cylindrical,  somewhat  rounded  cells,  varying  from 
1 to  2 microns  wide  to  4 to  6 microns  long,  close  together  in 
young  stages,  farther  apart  in  older.  Multiplication  by  fission, 
by  swarm  cells,  and  possibly  by  endospores,  with  sporelings 
developing  on  mother  filament.  Occurs  in  streams  receiving 
unpurified  sewage  and  in  the  effluent  of  industrial  wastes. 

Sphaerotilus  dichotomus  (Cohn)  em.  Migula  (1900  p.  1035) 
Synonyms: 

Cladothrix  dichotoma  Cohn,  1875 


15 


Chi arnydo thrix  sideroporus  Molish  1910* 

The  characters  of  S.  dicho teems  are  well  established, 
since  Migula  (1900,  p.  1026)  has  described  the  species  and 
since  Ellis  (a,  pp. 72-77)  has  figured  the  organisms  so  well  in 
plates  and  drawings. 

Migula  (1900,  p.  1036)  describes  S.  dicho tomus  as 
follows:  Thin,  fine  colorless  floes,  which  under  the  microscope 
appear  to  be  composed  of  dichotomous  threads.  The  threads  are 
surrounded  by  a thin,  firm  and  scarcely  visible,  sheath.  Migula 
and  Busgen  both  found  filaments  2 microns  thick.  Dichotomy  is 
the  result  of  a cell  breaking  out  of  the  sheath  but  still 
remaining  in  contact  and  covered  by  its  own  slime  sheath  and  by 
division  developing  a new  filament  which  appears  as  a branch  to 
the  mother  filament. 

Multiplication  takes  place  through  swarm  spores, 
which  appear  as  vegetative  cells  with  a cluster  of  flagella  near 
one  end.  They  leave  the  sheath  before  germination.  Found  very 
commonly  in  swamp  water,  occurs  in  iron  water  amongst  T.,epto  thrix 
ochraceae. 

As  noted  under  the  description  of  S.  natans  the  cells 
of  S.  dichotomus  are  very  close  together,  the  interval  between 
cells  being  hardly  visible  ( see  Plate  III,  Fig. 6). 

Ellis  (a,  p.76)  gives  the  following  characterization: 
In  a majority  of  cases,  5.  dichotomus  appears  as  isolated  long 
thin  filaments,  usually  in  great  numbers  attached  to  a common 
object.  The  cells  are  invisible  until  stained.  Growth  takes 


* 


Ellis,  p.  97,.  Considers  Chlamydo thrix  siderooorous 
s y no  ny mou  3 with  S . d i ch o tbW5 '.  ~~  


Molish  to  be 


17 


place  in  length  of  thread  only,  and,  as  the  filament  grows 
older  and  the  sheath  becomes  hardened  and  detached  from  the 
cells,  growth  results  in  the  disappearance  of  the  transverse 
septa  as  in  Crenothrix  . Ellis  represents  in  diagrams  and  shows 
by  plates  that  the  cells  of  S.  dichotomies  are  four  to  ten 
times  as  long  as  thick;  that  there  is  very  little  space  between 
cells;  that  the  sheath  is  very  thin,  and  that  dichotomy  may 
not  always  be  expected. 

Ellis  gives  three  methods  of  multiplication,  (1)  by  the 
liberation  of  thread  fragments,  (2)  by  the  liberation  of  re- 
juvenated single  cells  which  become  motile,  called  swarm  spores 
by  Migula,  and  (3)  by  the  formation  of  spiral  threads. 

Summary  of  Sphaerotilus  dicho tornus 

Filaments,  that  may  show  dichotomy,  composed  of 
rod-shaped  cells  approximately  2 microns  in  width  and  varying 
from  3 to  10  microns  long.  Sheath  thin,  firm,  visible.  Cells 
invisible  until  stained.  Occurs  in  thin,  fine,  colorless  floes, 
composed  of  a great  number  of  filaments  attached  to  a common 
object.  Growth  by  fission.  Multiplication  through  motile  swarm 
spores  which  are  ciliated  vegetative  cells  by  fragmentation  of 
the  filament,  or  the  formation  of  spiral  threads.  Occurs  in 
swamp  water  and  iron  water  where  Lento thrix  ochracea  is  found- 

Genus  CLONOTHRIX  Schorl er,  1904,  p.  68S 

Buchanan  (1918,  a,  p.  305^  gives  the  following  summary. 

Filaments  with  false  dichotomous  or  irregular 
branching  attached,  with  contrast  of  base  and  tip,  tapering  to  tip. 


18 


Sheath  always  present,  thin  on  young  filaments,  later  becoming 
thicker  and  encrusted  with  iron  or  manganese.  Multiplication  by 
small  non- mo tile  gonidiaof  spherical  form,  formed  from 
disc-shaped  cells  near  tip  by  longitudinal  division  or  rounding 
up. 

Number  of  species,  one. 

Clone thrix  fuse a Schorler 

Discovered  by  Schorler  in  the  water  works  of  Dresden 
and  described  in  1914,  but  has  not  been  found  elsewhere  since. 
Young  threads  are  2 to  3 microns  thick,  older  ones  5 to  7.  Cells 
measure  2 microns  in  thickness,  cylindrical,  some  longer  and 
some  shorter.  Cells  are  visible  without  staining. 

This  genus  perhaps  represents  a minor  local  variation 
of  the  common  type. 

3.  A Review  of  the  Taxonomy  of  the  Beggiatoaceae. 

The  order,  Thiobacteriales  is  represented  by  three 
families  (Buchanan  1918,  p.  461). 

Family  I.  Achromatiaceae  - Buchanan,  1916,  p.462 
Family  II.  Besa  atoaceae  - Migula,  1895,  p.  41 
Family  III.  R h o do b ac t er i a c e a e - Migula,  1900,  p.  1042 
Of  this  order  only  one  genus,  family  Beggiatoaceae 
was  observed  by  the  writer  in  the  flora  of  activated  sludge. 

Beggiatoaceae  Migula. 

Buchanan  (1918-p.463)  gives  a summary  of  the  characters 
of  the  family  and  a key  to  the  three  genera.  These  he  follows 

with  a description  of  each,  naming  a type  species. 


. 

. 


' 


19 


The  Beggiatoaceae  are  filamentous  bacteria,  usually 
showing  an  oscillating  motion  similar  to  Qscillatoria  not 
attached  and  no  differentiation  into  tip  and  base.  Cells  contain 
sulphur  granules.  Spore  formation  and  conidia  unknown. 

The  three  genera  are  : 

Cenus  1.  Thio thrix  - Winogradsky, 1888, p. 39 
Genus  2.  Be^eiatoa  - Trevisan,  1842,  p.76 
Genus  3.  Thionloca.-  Lauterborn, 1307, p. 238 

Beggiatoa  Trevisan 

Free,  sheathless  threads  composed  of  discoidal  cells. 
Motility  by  means  of  undulating  membrane  similar  to  Qscillatoria  . 
Reproduction  by  partial  fracture  and  subsequent  fragmentation. 
Widely  distributed,  occurs  in  situation  varying  from  potable 
waters  to  foul,  sulphur  springs  commonly,  fresh  water  and  marine. 
Number  of  species,  five. 

Type  of  species,  according  to  Buchanan,  Ber^iatoa  alba. 
The  species  of  Be.y iatoa  may  be  distinguished  by  the 
following  key  based  on  Migula's  description: 

Key  to  the  Species  of  Beggiatca 


1,  (4)  Cross  wall  indistinct  - 2 

2,  (3)  Filament  3.3  - 3.8  microns  thick B . alb  a 

3,  (2)  Filament  1.8  - 2.5  microns  thick B. Lentoformis 

4,  (.1)  Cells  (articulations)  of  the  filament  very  distinct  - 5 

5,  (8)  Found  in  salt  water  (marine)  situation  - 6 

6,  (7)  .f  ilament  more  than  three  microns  thick B.  mirabilis 

7,  (6)  Filament  less  than  three  microns  thick B.minema 

8,  (5)  Occurs  in  fresh  water  (swamps  or  sulphur  springs 

not  marine  B.  abachnoidae 


20 


Description  of  the  Species  of  Beggiatoa 
Note:  The  descriptions  following  are  based  on  Mi gula* 

(1900)  description  of  the  Be gel atoa.  particularly  in  the 
treatment  of  this  group.  References  are  not  so  complete  in 
detail  as  to  enable  one  to  arrive  at  all  of  the  sources. 
Nevertheless,  as  much  as  could  be  found  in  respect  to  dates  and 
particular  references,  has  been  included. 

Beggiatoa  alba  (Vaucher)  em.  Trevisan  1842 
Synonyms: 

Oscillator  la  alba  Vaucher  (?) 

Hy-rocrocis  vandellii  Meneghini  (?) 

Beggiatoa  punctata  Trevisan,  1842,  p.56 
Filament  without  recognizable  cross  walls,  3.3  to 

3.8  microns  thick.  Occurs  in  pale  slimy  masses  in  foul  water, 
sulphur  springs  and  swamps.  Most  common  species. 

Beggiatoa  1 eutoformis  (Meneghini)  Trevisan  1842 
Syno nyms: 

Qscillatoria  leptof itiformis  Meneghini 

Filament  without  recognizable  cross  walls,  very  thin, 

1.8  to  2.5  microns  thick.  Occurs  in  great  quantities  in  pale 
and  slimy  masses  in  sulphur  springs,  also  in  swamps  and  waste 
waters.  Not  as  common  as  B . alba  but  found  with  it. 

The  only  difference  between  the  descriptions  of  B . alba 
and  B.  leptoformis  is  one  of  size.  For  all  practical  purposes 

the  two  species  are  identical  especially  since  they  occur 
together . 


21 


Beggiatoa  mirabllls  Cohn  1865,  p.  81 

Filaments  13  microns  thick,  articulated  and  variously 
curved  and  tangled,  with  rounded  ends.  Length  of  cells  1/2 
times  width.  Filaments  associated  with  a snow-white  slime. 
Marine. 

Be^iatoa  minema  Warming  (?) 

Similar  to  B.  leptoformis.  except  that  the  filaments 
are  articulated  (Compound  of  cells)  1.8  to  2 microns  thick. 
Length  of  cells  1/2  times  width.  Longest  threads  40  microns. 
Marine. 

The  same  can  be  said  of  these  two  marine  forms  as  was 
said  of  the  two  previous  fresh  water  forms:  the  descriptions  are 
quite  identical  except  as  to  the  size  of  the  filaments. 

Be-?,£iatoa  arachnoidae  Rabenhorst,  p.  94 
Synonyms: 

Oscillator i a arachnoidae  ( ? ) 

Beg-viatoa  pellucida.  Cohn  (?) 

Articulated  filaments,  5 to  6.5  microns  thick,  cells 
length  cells  1 to  1.2  times  width.  Not  marine.  Occurs  in 
swamps  and  sulphur  springs. 

Summary  of  Bergiatoa. 

There  are  two  distinct  types  of  B egsl ato a.  One  type 
articulated,  the  other  without  visible  cross  walls.  The  former 
occurs  both  in  fresh  and  salt  waters,  while  the  latter  has  been 
found  only  in  fresh  water. 


22 


III 

MATERIAL  AND  METHODS 

The  microscopical  observations  with  which  this  paper 
is  concerned  were  made  from  November  15th  to  December  29th,  1921. 
It  was  desired  (1)  to  trace  the  development  of  the  sludge  from 
raw  sewage  to  a climax  sludge,  (2)  to  observe  the  evolution  of 
forms  that  takes  place,  and  (3)  to  identify  the  types  present. 

As  before  stated,  the  present  study  is  concerned  with  the 
filamentous  forms  only. 

Samples  of  sludge  taken  from  the  up  cast  well  of  the 
aeration  chamber  were  brought  in  daily,  excepting  Sunday. 
Observations  were  made  on  these  samples  and  permanent  slides 
prepared  from  each. 

Counts  of  various  forms  including  zoogloeal  masses, 
were  made.  The  results  are  reported  in  an  unpublished  article. 

In  as  much  as  we  are  concerned  with  Beggiatoa  here  the  method 
used  in  counting  will  be  described.  The  one  cubic  centimeter 
rectangular  cell  was  filled  with  sludge,  and  counts  were  made  of 
ten  fields  one  millimeter  square.  Estimations  of  forms  or  units 
present  per  cc.  were  based  on  these  ten  counts.  (Generally,  two 
mounts  'were  made  with  five  fields  counted  from  each.) 

In  the  preparation  of  permanent  slides,  a few  drops 
of  sludge  were  evaporated  on  the  slide  and  fixed  by  heating. 
Carbol  fuchsin  and  gentain  violet  were  used  to  stain  different 
areas  of  the  same  slide. 

In  as  much  as  the  quantitative  observations  on  the 


filamentous  forms  can  be  determined  only  relatively,  direct 
count  being  impossible,  three  terms  — f ew , common,  abu no. ant , are 


. 


- 

. 


. 


t 


■ 


■ 


■ 


23 


used  by  the  writer  to  describe  the  quantitative  relationships. 

Low  power  magnification  as  used  in  the  observations  indicates 
a magnification  of  100  diameters. 

IV. 

OBSERVATIONS 

The  characteristic  appearance  presented  by  an  unstained 
field  under  lower  power  magnification  is  as  follows:  One  to  three 

Bep.;giatoa  filaments  may  be  in  the  field,  as  many  or  more 
peritrichs,  a few  hypotrichs,  and  perhaps  a nematode,  - these, 
moving  around  amongst  the  masses  of  sponge-like  material 
composed  of  a network  of  filamentous  forms,  the  so-called  iron 
bacteria. 

Sludge  has  been  described  as  amorphous  colloidal 
material.  The  writer  would  describe  climax  sludge  material  as 
consisting  primarily  of  a network  of  filamentous  bacteria,  a 
delicate  sponge-like  mass,  intersperced  with  enmeshed  amorphous 
material.  In  the  amorphous  material  can  be  found  representatives 
of  every  morphological  type  of  bacteria,  with  the  zoogloeal 
growth  habit  common.  One  is  impressed  by  the  mass  of  filaments 
that  compose  the  foundation  of  the  sludge,  and  the  writer  does 
not  doubt  that  they  are  organisms  of  greatest  sanitary  significance. 

Just  what  these  filamentous  forms  are  and  their 
prominence  and  time  of  appearance  in  the  formation  of  the  sludge 
will  be  the  subject  matter  of  the  following  paragraphs. 

In  the  consideration  of  the  filamentous  bacteria  it 
is  well  to  keep  in  mind  that  all  of  the  descriptions  given  in  the 
foresoins:  discussion  are  of  European  xorms,  with  one  minor  excep— 

O O 

tion.  We  should  expect  to  find  such  widely  distributed  forms 


24 

fairly  constant  in  morphological  characters  the  world  over. 

On  the  other  hand,  observations  have  been  more  or  less  fragmen- 
tary in  many  cases,  and  we  may  find  upon  Continued  observation 
that  forms  believed  to  be  different  are  variations  of  a common 
type. 

Occurrence  of  Beggiatoa. 

The  occurrence  of  Beggiatoa  was  fairly  constant 
throughout  the  period  of  observation,  especially  after  the 
mature  sludge  was  developed.  A few  filaments  were  observed 
during  the  earlier  days.  During  the  period  of  climax  sludge 
the  counts  per  cubic  centimeter  varied  from  100  to  5500. 

Beg- iatoa  was  easily  recognized  because  of  the  motility  and 
easily  counted  because  of  the  small  number  as  compared  with 
other  filamentous  forms. 

Filaments  present  varied  from  1.5  microns  to  3.5 
microns  in  thickness,  all  without  visible  cross  walls.  The 
larger  form  described  as  B.  alba  was  the  most  abundant,  occurring 
in  a proportion  of  about  three  to  one  with  the  smaller  form 
described  as  B.  arachnoidae. 

Occurrence  of  Sphaerotilus  dichotomus 

The  first  appearance  of  S,  dichotomus  was  noted  on 
November  28th,  thirteen  days  after  the  beginning  from  raw  sewage. 
While  making  counts  under  low  power  branching  colorless  filaments 
were  observed.  Stained  mounts  revealed  the  character  of  the 
filaments  a^  represented  in  Plate  III,  Fig.  6.  Such  filaments 
were  very  few  until  December  12th  when  they  became  common,  but 


■ i */■  f 

■ 


25 


never  abundant.  S.  dichotomus  and  Beggiatoa  alba  occurred 
in  approximately  equal  proportions. 

Occurrence  of  Crenothrlx  polyspora 

Crenothrix  in  the  immature  filaments  was  common  in  the 
observations  made  on  the  first  samples  taken.  Rapid  increase 
occurred  so  that  at  all  times  the  typical  Crenothrix  filament 
was  in  predominance.  After  fifteen  days  operation  the  appearance 
of  the  sludge  in  the  stained  mount  was  characteristic  of  that 
which  continued  throughout  the  rest  of  the  period,  with  a 
possible  increase  in  the  percentage  of  filamentous  forms. 
Crenothrix  as  indicated,  predominates  over  all  other  forms 
present. 

The  writer  wishes  to  define  what  he  has  included 
under  the  name  Crenothrix.  Some  of  the  facts  necessary  in  the 
consideration  of  the  filamentous  bacteria  have  been  enumerated 
in  a previous  paragraph.  Already  many  forms  have  been  recog- 
nized as  variants  and  have  been  included  as  synonyms,  perhaps 
in  sludge  different  varieties  are  represented,  and,  indeed, 
had  the  several  growth  habits  and  morphological  types  been 
observed  by  themselves,  they  might  have  been  mistaken  for  new 
species,  or  possibly  new  genera.  But  rather  than  make  the  error 
which  earlier  observers  have  made  and  cause  still  more  confusion, 
the  several  variants  of  a common  type  are  included  under  the 
genus  Crenothrix  because  the  description  of  that  genus  is  broad 


and  quite  definitely  accepted. 

In  as  much  as  in  some  sanitary  discussions  ^.Johnson,  1914 


• 

- 

■ 

26 


the  name  Sphaerotilus  natans  is  used  in  the  description  of  the 
flora,  the  writer  has  included  the  description  of  the  genus 
in  the  foregoing  consideration.  Perhaps  the  genus  has  not  been 
sufficiently  described  to  bear  consideration  or  comparison, 
if  not,  then,  with  Ellis,  we  need  not  consider  it  a definitely 
established  genus.  Ellis  believes  (a,  p.93)  that  the  generic 
and  even  specific  distinctness  of  Sphaerotilus  natans  is 
doubtful.  The  writer  believes  the  observations  which  follow 

will  corroborate  his  statement.  (This  does  not  invalidate  the 
entire  genus,  however ). 

In  the  first  place,  we  note  that  the  description  of 
Sphaerotilus  natans  and  Crenothrix  polyspora  are  quite  similar. 
There  are  three  differences  noted: 

1.  S . net ans  apparently  has  less  power  to  precipitate 
iron  oxide  than  Crenothrix . 

2.  The  habit  growth  of  S.  natans  is  in  parallel 

strands . 

3.  There  is  a difference  in  method  of  multiplication. 

In  case  of  the  first  difference  it  is  not  an  absolute 
difference.  Not  all  Crenothrix  filaments  are  iron  incrusting, 
and  as  indicated  some  Sphaerotilus  natans  may  be  iron  incrusting. 

Secondly,  it  is  highly  possible  for  Crenothrix  to 
simulate  the  growth  habit  which  characterized  Sphaerotilus  natans. 
If  a group  of  filaments  with  gelatinous  sheaths  were  pressed 
together  as  they  developed,  it  would  be  very  natural  for  them  to 
maintain  such  an  or ganization.  Such  growth  habits  have  occurred 
in  activated  sludge  composed  of  filaments  which  in  the  writer's 


27 


judgment  could  be  classified  only  as  Creno thrix.  The  writer 
has  observed  similar  growth  habits  in  great  abundance  in 
sludge  fed  by  starch  wastes. 

The  question  of  multiplication  will  be  considered 
under  variation. 

The  most  conclusive  evidence  that  the  writer  can  find 
for  the  identity  of  Creno thrix  and  Sphaerotilus  natans  is  the 
fact  that  no  author  has  ever  described  both  of  them  as  occurring 
together,  but  when  described  they  came  from  identical  situations. 
If  they  occur  in  identical  situations  why  have  they  not  been 
described  together?  In  fact,  an  author  who  recognizes  one  in 
his  observations  never  mentions  the  other.  In  one  case, 
Sphaerotilus  natans  was  found  in  effluent  from  industrial  wastes. 
That  would  indicate  that  what  was  called  S . natans  due,  to 
environment,  might  be  merely  a variant  of  Creno  thrix . In  fact, 
the  original  form  of  Cr eno thr ix  described  is,  perhaps  a variant 
of  a common  type,  as  shall  be  noted  in  the  following  paragraphs. 
If  such  be  the  case,  we  might  expect  a great  variation  in  an 
environment  such  as  is  encountered  in  the  activated  sludge. 

Variations  from  the  Common  Type. 

(1)  Ellis  observes  that  we  must  not  always  expect  to 

find  Creno thrix  assuming  the  "trumpet”  shape  at  the  free  end, 
a character  which  is  so  often  used  to  describe  it.  The  writer  has 
never  found  it  assuming  such  a form,  neither  have  other 
investigators  of  American  forms  recorded  such  forms. 

Evidently  the  method  of  multiplication  which  causes 
the  formation  of  the  "trumpet"  is  uncommon  to  American  forms. 


28 

On  the  other  hand,  the  type  of  multiplication  as  observed 
by  Jackson  (1302)  is  quite  common  under  certain  conditions. 

The  cells  of  the  filament  break  up  into  a number  of  small 
spores  causing  a disc-like  appearance  through  the  filament. 

Masses  of  Cr eno thrix  filaments  have  been  found  in  such  a state 
in  a minature  experimental  sewage  disposal  plant  in  the  labora- 
tories of  the  State  Water  Survey. 

These  same  filaments  show ed  a readiness  of  fragmentation 
into  segments  containing  10  to  15  of  the  disc-like  cells.  Such 
fragmentation  is  ascribed  to  S,  dichotomies  (Ellis,  a,p.88). 

Whether  such  fragments  possess  motility  has  not  been  determined. 
That  these  fragments  are  not  S.  dichotomus  is  certain  because 
they  are  visible  unstained. 

(2)  The  description  of  Creno thrix  is  quite  broad, 
includingf i laments  of  both  discoidal  and  cylindrical  cells. 
Filaments  made  up  of  discoidal  cells  characteristically  Creno  thrix 
occur  in  abundance  throughout  the  activated  sludge.  These 
filaments  range  from  the  very  young  and  immature  filaments,  .5 
to  .75  microns  in  thickness,  to  the  older,  2 micron  in  thickness. 
They  occur  quite  characteristically  in  parallel  heavy  thick 
masses  (Plate  II,  Fig. 4),  although  they  occur  very  commonly  as 
single  filaments. 

The  most  striking  variation  is  shown  in  Plate  II,  Fig. 

3,  in  which  a filament  composed  of  cylindrical  cells  has  divided 
up  into  discoidal  cells  leaving  the  interval  between  the 
original  rods  plainly  visible.  In  filaments  from  2 to  3 mm. 

long,  it  is  possible  to  find  cells  at  one  end  normally  rod-shaped. 


29 


Toward  the  center  of  the  filaments  many  refractile  areas 
appear  through  the  length  of  the  cells  (which  is  of  common 
occurrence  just  before  or  during  the  process  of  assuming  the 
discoidal  form).  At  the  opposite  extreme,  the  rods  have  become 
a segment  of  discs.  Plate  I,  Fig.l  in  the  upper  left  hand 
corner  3hows  a short  filament  in  which  the  refractile  areas 
occur . 

Eidam,  (1376,  p.133)  already  referred  to  in  the  con- 
sideration of  Sphaerotllus  natans  found  such  refractile  areas 
and  called  them  what  would  be  interpreted  as  endospores.  He 
describes  a condition  which  the  writer  found  and  reproduced  in 
Plate  I,  fig.  2.  He  describes  the  spores  escaping  through  the 
gelatinous  matrix  and  germinating  on  the  mother  filament.  Of 
course,  spores  may  be  attracted  to,  or  become  attached  to  the 
surface  of  the  filament  from  the  exterior,  but  it  is  more 
reasonable  to  assume  that  the  multitude  of  short  sporelings 
as  shown  in  Plate  I,  Fig. 2 originated  from  the  cells  within  the 
filament  rather  than  that  they  are  merely  gathered  from  the 
surrounding  medium.  The  fact  that  refractile  areas  appear  in  the 
cells  and  that  not  all  filaments  are  equally  covered  with 
sporelings,  some  having  none,  others  many,  lends  evidence  toward 
that  assumption. 

Eidam  believed  the  form  to  be  Sphaerotilus  natans 
since  it  answered  that  description.  But,  as  far  as  morphological 
characters  are  concerned,  it  answers  the  description  of  Ore no thri x. 
We  have  already  described  a condition  very  similar  under  (1)  in 
the  reference  made  to  Jackson  (1902).  Even  in  Plate  I,  Fig.l, 


■ 


- 


. 


- 


30 

a few  short  sporelings  appear,  but,  in  addition,  many  long 
sporelings  which  give  outline  to  the  invisible  sheath,  of 
course,  the  origin  of  the  sporeling  could,  be  determined  certainly 
if  one  could  isolate  the  forms  in  pure  culture.  Until  such  a 
technique  is  developed  one  can  only  speculate  on  the  matter. 

Again,  the  rods  of  the  filaments  shewn  in  Plate  III, 

Fig.  5,  seem  identical  with  those  figured  by  Migula  (1900, 

Plate  XVIII,  Fig. 8).  The  writer  found  such  filaments  growing 
in  the  parallel  habit  as  shown  by  Migula  (1900,  Plate  XVIII, Fig. 7). 
But  on  comparison,  Plate  ill  is  identical  with  the  forms 
represented  in  Plates  1 and  II.  There  may  be  a difference  in 
development  in  the  particular  filaments  shown,  but  the  morpho- 
logical characters  are  identical,  as  well  as  the  refractile 
areas  common  to  both. 

Sphaero tllus  has  been  described  as  forming  swarm 
cells.  This  character  has  been  used  to  separate  this  genus 
from  Crenothrix.  Since  these  forms  have  not  been  isolated  in 
pure  culture  it  is  difficult  to  determine  the  origin  of  swarm 
spores.  In  the  second  place,  granting  the  formation  of  swarm 
spores  it  cannot  be  said  that  such  a character  is  less  likely 
to  be  a variation  from  the  common  method  than  the  other  mode  of 
multiplication  cited. 

By  way  of  summary  of  this  comparison,  then  it  has 
been  shown  that  Sphaero tilus  natans  is  synonymous  with  Cr enc thri x 
polyspora,  or  that  they  are  both  variants  of  a common  type; 
that  the  type  represented  is  quite  subject  to  its  environment, 
assuming  various  modifications  of  a common  type.  (Fig.  1, 2, 3, 4, 5 ) , 


31 


V. 

SUMMARY 

1.  Filamentous  forms  increase  from  "few"  to  "abundant" 
during  the  first  ten  to  fifteen  days  of  sludge  formation. 
Following  the  first  period  of  ten  to  fifteen  days,  under  the 
particular  conditions,  the  sludge  continued  primarily  as  a 
network  of  filaments. 

2.  Creno thr ix  is  present  at  all  times  and  is  the  pre- 
dominating type  of  organism. 

Filaments  of  the  type  Creno thrix  are  subject  to 
great  variation.  Perhaps  some  of  the  variants  deserve  -che 
designation  of  species,  but,  in  as  much  as  they  are  without  a 
doubt  due  to  immediate  environmental  influences,  they  should  be 
considered  merely  as  growth  habits,  at  least  until  isolation  in 
pure  culture  is  accomplished. 

Sporelings,  short  and  long,  occurred  commonly  in 
connection  with  Creno  thrix . never  in  connection  with  Sphaero tilus 
aichotomus  and,  therefore,  they  possibly  originate  from  spores 
produced  by  filaments  of  the  Creno thrix  type. 

3.  Sphaero tilus  dichotomus  (Syn:  Cladothrix  dichotoma 
Cohn)  appeared  thirteen  days  after  the  beginning  from  raw 

sewage  and  remained  a constantly  occurring  form  being  "common" 
after  December  12th. 

4.  Two  species,  one  type,  of  Be^giatoa  were  observed 
sparingly  during  the  formation  period  of  the  sludge  and  became 
common  in  the  climax  sludge,  occurring  approximately  in  same 
proportion  as  Sphaero tilus  dichotorms. 


32 


VI. 

ACKNOWLEDGEMENT 

The  writer  wishes  to  thank  Dr . A.  M. Buswell 
(Chief  of  the  State  Water  Survey)  for  the  opportunity 
to  conduct  this  investigation  and  for  the  U3e  of 
laboratories  and  materials.  To  Dr.  Stella  M. Hague, 
under  whose  supervision  this  investigation  was  con- 
ducted, the  writer  is  indebted  for  invaluable  criticisms 
and  suggestions. 


33 


VII. 

BIBLIOGRAPHY. 

Benecke,  Wilhelm,  1912,  Ban  and  Leben  der  Bakterien 

Buchanan,  R.  E.,  ISIS,  (a)  Studies  in  the  Classification  and 
nomenclature  of  the  Bacteria.  Journal  of  Bacteriology, 

Vol.III,  pp.  301-306. 

(b)  Same  title,  Journal  of  Bacteriology,  Vol.III,  pp. 481-474 

Cohn,  Ferdinand,  18  65,  Hedwigia 

1870,  Unter suchungen  uber  Bakterien,  Beit.z. 

Biol.  d.  Pflanzen,  Vol.  1,  h.l, Breslau. 

1875,  U nter sue hungen  uber  Bakterien,  II,  Beit.z. 
Biol.  d.  Pflanzen,  Vol.  1,  h.3,  Breslau. 

Cox,  Charles  R.,  1921,  Non  Bacterial  Population  of  Sewage  Trickling 
Filters.  Eng.  News  Record,  Vol.  87,  18,  p.  720. 

Ehrenberg,  C.G.,  1838,  The  Infusionstheierchen  alvollkommende 
Organ! smen.  Le ip zing. 

Eidam,  1876,  Ueber  die  Entwickelung  des  Sphaerotilus 

natans.  Jahres  ber  d.  Schlesisch.  Geselisch.  f,  vaterl. 

Kultur,  p.  133. 

Ellis,  David,  1907,  (a)  Proc.  Roy.  Soc.  Edinburg,  vol.27,p.21 

1907,  (b)  A Contribution  to  our  Knowledge  of  the 
Thread  Bacteria.  Centrabl.  f.  Bakteriol.  Abt.  2,  Vol.  19, 
p.  503. 

(a)  The  iron  Bacteria,  (Pub.  by  F.  Stoakes 
& Co.  bearing  no  date.) 

Enlows,  Ella,  M. A, 1920,  The  Generic  Names  of  Bacteria.  U.S.P.H. 
Service,  Hygienic  Lab.  Bull. 121. 

Griffith,  J.W. , 1853,  On  Gallionella  ferruginea.  Ann  and  Magaz. 
of  Nat. Hist. London  Ser.2,  Vol.  12,  p.438. 

Hommon,  Harry  B.,  1918,  The  Treatment  and  Disposal  of  Strawboard 
Waste.  Public  Health  Bulletin,  No.  97,  pp. 58-69. 

Jackson,  D.D.,  1902,  A New  Species  of  Crenothrix.  Trans. Am.  Hie. 
Society,  Vol  23,  p.33. 

Johnson,  J.W.Haigh,  1914,  A . Contribution  to  the  Biology  of  Sewage. 
Jour,  Ec,  Biology,  Vol.  9,  pp. 105-124,  127-134, 

Keutzing,  F.  T. , 1833,  Linnaea,  Berlin,  Vol, 8,  pp.19,385 

1843,  Phytolgia  Generalis,  Leipzing.  p.198 


Bib.  -2 


Lauterborn,  Root.,  1307,  Ein  neue  Gattung  der  Scwef elbakter ien. 
Ber.  d.d.bot.  Gesellschaf t . , vol. 25,  pp.23S-242 

Migula,  W. , 1895,  Schizomycetes,  in  Engler  and  prantl.  Natur- 
lichen  Pflanzenf amilien,  p.  20. 

Migula,  W. , 1900,  System  der  Bakterien,  Berlin. 

Molish,  H. , 1892,  Die  Pflanzen  in  ihrer  Beziehungen,  zum  Eisen. 
Jena. 

1310,  Die  Eisenbakter ien.  Jena 
New  jersey  Ag.  Exten.  Bull.  352. 

Parkinson,  1319,  Parkinson  and  Bell. Insect  Life  on  Sewage  Filters. 
San.  Pub. Co . Ltd. London. 

Porter,  J. Edward,  1921,  A Bibliography  on  "The  Activated  Sludge 
Process  of  Sewage  Treatment".  Gen. Filtration  Co.  Inc. 
Rochester,  N.  Y. 

Rabenhorst,  Flora  Europ.  Alg. , Bd.II,  p.94. 

Russel  and  Bartow,  1916,  Bacteriological  Study  of  Sewage  Purifi- 
cation by  Aeration,  Water  Survey  Series,  Illinois,  No.  13, 
pp. 348- 358 . 

Schorler,  1904,  Beitrage  zur  Kenntniss  der  Eisenbakteri en. 
Centrabl.  f.  Bakteriol.  Abt.2,  Vol.  12,  p.  689. 

State  Board  of  Health  of  Mass,  1912,  The  44th  Annual  Report. 

Trevisan,  V.,  1842,  Prospetto  della  Flora  Eugana,  p.  56. 

1879,  Prime  linee  d’  introduzione,  alio  studio  dei 
Batter j.  italiani.  Rendiconti.  Reale  Instituto  Lombardo  di 
Scienze  e letters  IV.  Ser.  2,  Vol.  12,  p.  133-151. 

1885,  Caratteri  di  alcumi  nuovi  generi  di  batteroi- 
acee.  Atti  della  A ccademia  Fiso-Medico-Stati3tica,  in  Milano. 
Ser.  4,  Vol.  3,  pp. 92-107. 

Warming,  Om  nogle  ved  Danmarks  Kyster  levende  Bakterier.p.  356. 

Winogradsky,  1888,  Fur  Morphologie  und  Physiologie  der 

Sohwef elbacterien  Beit..z.  Morph,  und  Physio. d. Bakterien  h.l. 


. 


34 

VIII. 

PLATES  A ITT  DESCRIPTIONS 

The  microphotographs  composing  the  following  plates 
were  taken  by  Arthur  Granville  Eldridge,  Instructor  in 
Photography  in  the  Laboratories  at  the  University  of  Illinois 
from  slides  prepared  by  the  writer. 


Fig.  1. 


Crenothrix  polyspora. 

Main  filament,  a semi-mature  filament  of 
The  sheath  is  well  outlined  by  short  and 
lings. 

Arrow  indicates  short  Crenothrix  filament 
refractile  areas. 


Creno thr ix. 
long  spore- 
showing 


Fig.  2. 


Fig.  3. 


Crenothrix  polyspora. 

Shows  filaments  heavily  decorated  with  sporelings. 
Arrow  indicates  a filament  of  Sphaerotilus  dichotomus. 
Near  edges  of  dark  masses  spores  and  coccoid  bacteria 
are  shown.  The  dark  areas  are  chiefly  such  forms  in 
connection  with  the  filaments  and  amorphous  material. 


Crenothrix  polyspora. 

Figure  shows  normal  filament 


in  which  the  individual 


rods  have  divided  up  into  disc-like 


segments. 


F i g.  4 . 


Fig.  5. 


Same  as  above,  but  showing  a common  growth  habit  of 
younger  filaments.  The  filaments  of  this  mass 
apparently  developed  in  the  disc-like  form  at  the 
origin  of  the  filament.  The  mass  is  r o thick  that  a 
focus  can  be  obtained  only  on  one  level.  Arrows 
indicate  place  at  which  structure  is  best  represented, 
Thi3  is  the  commonest  form  appearing  throughout  the 
sludge.  Such  forms  are  distributed  through  the  heavy 


masses  m 


greater 


numbers  than  any  other  type. 


Crenothrix  polyspora. 

Two  main  filaments  are  of  same  type  shown  in  Plate  I., 
but  in  a different  state  of  development.  Sheath  not 
recognizable  except  as  suggested  by  few  particles 
which  mark  the  sheath  limits.  Upper  filament  shows 
fission. 


Fig.  6.  Sphaerotilus  dichotomus. 

Arrow  indicates  the  part  of  filament  best  in  focus 
Very  thin  sheath,  long  rods,  with  short  interval 


between.  Contrast  with 


Fig. 


1. 


Fig.  7. 


A 


zoogloeal 


habit  of  growth  common  in  the 


sludge.  As  indicated  by  the  forming  filament. 


activated 
this 


is  perhaps  a zoogloeal  state  a^ 
form  that  is  evidently  related 
Plate  II,  Fig.  4. 


sumed  by  a filamentous 
to  the  type  shown  in 


PLATE  I 


*ig.  1 

x 75C 


Fi  g.  2 

x 500 


PLATE  II 


Fig.  3 
x 500 


i'ig.  4 
x 500 


PLATE  III 


Fig.  5 
x 500 


Fig.  6 
x 500 


PLATE  IV 


